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Working Group on New TB Drugs

Stop TB Partnership

Meetings

"Essentiality of PZA" Workshop 2011

May 31 - June 1, 2011 | Bethesda, Maryland

Meeting Overview

DAY 1 May 31, 2011 7:00 p.m. - 10:00 p.m.

Working Dinner with Special Presentations by Jacques Grosset
and Oren Zimhony
Bethesda Marriott | 6711 Democracy Boulevard | Bethesda, Maryland 20817

DAY 2 June 1, 2011 8:15 a.m. to 4:30 p.m.

Essentiality of PZA Workshop
NIH/NIAID | 6610 Rockledge Drive, Room 4008 | Bethesda, Maryland 20817-1811

May 31, 2011Day 1 Dinner
Special Presentation Oren Zimhony

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  • The advantage of pyrazinamide(PZA) therapy in rendering tissues of tuberculosis infected mouse to be "non-culturable" lead to its designation as "sterilizing" agent which was translated clinically to lasting cure for patients at the shortest course of therapy. However, PZA activity in vitro is relatively poor and non-bactericidal. The explanation for this apparent paradox should be sought in enhancement of its mechanism of killing in-vitro upon in vivo conditions (such as activated macrophages). Since PZA is mainly effective when given in the early phase of tuberculosis therapy and all culture tests methods for PZA susceptibility advocate and rely on the usage of replicating bacilli, PZA like other drugs should be studied with replicating bacilli.
  • PZA activity against mycobacteria depends on conversion to pyrazinoic acid (PA) by pyrazinamidase (PZAase). M. tuberculosis is susceptible to PZA only at acidified medium. The unique activity of PZA against M. tuberculosis amongst mycobacteria is due to an inefficient energy dependent efflux. The three factors conversion to PA (indispensible) acidic pH and deficient efflux combine, and result in "huge" intracellular accumulation of PA, which did not affect the intracellular pH.
  • PZA analogs were synthesized; their activity does not rely on PZAase conversion to PA. Many of the different PZA analogs have much higher potency against M. tuberculosis and also an expanded spectrum against other species of mycobacteria. PZA and its analogs likely share the same cellular site between mycobacteria species and PZA derivatives as such interaction is highly specific and essential cellular functions are not redundant.
  • The requirement of acidic pH for PZA activity can be markedly diminished by increased PZAse activity (converting PZA resistant M. smegmatis to PZA susceptible), by inhibition of efflux such as by reserpine and the analogs 5 chloro PZA and n-propyl PA, are active in neutral pH. The analog 5-Cl-PA is a stronger acid yet a weaker antimycobacterial agent than PA. Thus, acidity of the medium, merely a condition for PA accumulation, cannot have a role for the killing effect of PZA and intracellular acidification by PA is unlikely either. The dependence of PZA activity upon PA accumulation suggests that it affects an intracellular function.
  • PA and PZA analogs at equivalent dose (folds of the minimal inhibitory concentration, MIC) have similar antimycobacterial effect or killing curve. The antimycobacterial effect of PA and PZA analogs in replicating bacilli correlates with inhibition of fatty acid biosynthesis mediated by the multi-domain ,multifunctional enzyme fatty acid synthase I ( FAS I). These compounds inhibit FAS I in vitro (albeit at high concentration for PA and PZA), and were found to interact with FAS I enzyme in saturated transfer (STD)NMR studies.
June 1, 2011Day 2 Session 1
What Mice Tell Us About the Essentiality of PZA Eric Nuermberger

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  • When used as part of regimens not containing isoniazid, PZA contributes sterilizing potential that extends beyond the first 2 months of treatment in murine models.
  • Combinations containing a potent fluoroquinolone, an injectable and PZA may be capable of shortening the duration of MDR-TB treatment to 9 months.
  • PZA improves the activity of virtually all new drugs in clinical development in murine models of TB.
  • In vitro and in vivo data suggest there may be mutual antagonism between isoniazid and pyrazinamide.
Nicole Ammerman Presentation Video Does pyrazinamide have a host-directed mechanism of action? Nicole Ammerman

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  • In our experimental system, the addition of pyrazinamide, nicotinamide, pyrazinoic acid, nicotinic acid or isoniazid to uninfected J774 cells did not induce a proinflammatory cytokine response, and the addition of these compounds to M. tuberculosis-infected J774 cells did not alter cytokine secretion levels or patterns.
  • In the same experimental system, very few gene expression changes were observed with the addition of the above-stated drugs to either uninfected or infected J774 cells.
  • Administration of PZA 25 days by oral gavage was associated with a proinflammatory cytokine response in the lungs of both uninfected and M. tuberculosis-infected C3HeB/FeJ mice.
Session 2
Susana Mendez Presentation Pyrazinamide treatment prevents the clinical signs of cutaneous leishmaniasis Susana Mendez

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  • Think outside the box: PZA may have activity against other microorganisms.
  • Leishmaniasis is a chronic, emerging parasitic disease caused by the protozoan parasite Leishmania. Clinical signs range from mild cutaneous lesions to systemic disease leading to death.
  • PZA has activity against Leishmania in vivo in both models of cutaneous and visceral disease.
  • Some PZA analogs (i.e. 5-chloro and 5-fluoro) had greater activity than the parental compound 5.The target for PZA in Leishmania is unknown, but our two main hypotheses are that the drug inhibits the activity of either microsomal elongases or sirtuins in this parasite.
David Russell Presentation What do we know about the intracellular environment experienced by M. tuberculosis? David Russell

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  • M.tb inhabits an intracellular environment close to pH6.4 that is relatively benign with respect to hydrolytic activity.
  • This environment places certain physiologic and metabolic requirements on the bacterium.
  • The bacterium appears to shift its carbon metabolism to rely on fatty acids, lipids and steroids for survival.
  • We will never understand the activity of drugs like PZA if we cannot appreciate or reproduce this environment.
  • In consequence all drug discovery, or target discovery, programs need to incorporate this into their assays.
Catherine Vilcheze Presentation nad biosynthetic mutants of Mycobacterium tuberculosis: a tool to elucidate PZA action Catherine Vilcheze & William Jacobs

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  • The mode of action of PZA is still mostly unknown.
  • PZA as a structural analog of nicotinamide could interfere with NAD+ salvage pathway. which uses nicotinamide to synthesize NAD+.
  • PZA could enter the NAD+ salvage pathway and either form a PZA-analog of a NAD+ metabolite and inhibit NAD+ biosynthesis or form a PZA-analog of NAD+ that could inhibit enzymatic reactions using NAD+ as cofactor.
  • Since PZA has been shown to have an effect on NAD+ concentration in eukaryotes, these hypotheses deserve to be studied.
Session 3
Anthony Baughn presentation video In search of parsimony: An alternative hypothesis for the mode of action of pyrazinamide Anthony Baughn

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  • The mode of antimycobacterial action for pyrazinamide has yet to be proven.
  • Pyrazinoic acid, the supposed active form of pyrazinamide, chelates divalent metal ions such as iron and manganese.
  • Metal sequestration is a potential alternate or additional mode of antimycobacterial action for pyrazinoic acid.
Pyrazinamide, sterilizing drug only? Jacques Grosset

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  • Like rifampin, pyrazinamide is a sterilizing drug that accelerates the killing of "persisters" and is responsible with rifampin for the shortening of the TB treatment duration from 18 months to 6 months.
  • Pyrazinamide is often considered bactericidal against actively replicating tubercle bacilli.
  • In fact, we demonstrated that pyrazinamide has clear-cut activity against nonreplicating bacilli but no activity at all against actively replicating bacilli in the nude mouse model.
  • These findings support the hypothesis that accumulation of toxic products of pyrazinamide metabolism within nonreplicating bacilli is the mechanism by which pyrazinamide kills tubercle bacilli.
  • These findings raise the important question of why pyrazinamide is used during the initial 2-month phase of TB treatment and not during the continuation phase.
Efficacy of PZA administered as a single drug in various TB mouse infection models (GKO, Balb/c and Kramnik) Anne Lenaerts

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Session 4
Ying Zhang Video Presentation The Roller Coaster of PZA Ying Zhang

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  • PZA is a frontline drug that has unique sterilizing activity involved in shortening treatment of TB and MDR-TB, as it kills persister TB bacilli that are not killed by other TB drugs and is essential or irreplaceable in any new drug combination for treatment of TB.
  • PZA is a paradoxical TB drug that has no activity against TB bacilli in vitro at neutral pH when bacteria are growing but is only active at acid pH. The activity of PZA is enhanced under stress conditions such as starvation, hypoxia, energy inhibitors, etc, and is completely opposite to common antibiotics.
  • We identified a new target of PZA RpsA that binds to POA where overexpression of RpsA conferred 5-fold PZA resistance in MTB and a low level PZA-resistant M. tuberculosis DHM444 without pncA mutations carried an alanine deletion due to 3-bp (ΔGCC)missing in the C-terminus of RpsA.
  • The active form of PZA, pyrazinoic acid (POA), at therapeutically relevant concentrations (50-100 ug/ml), inhibited the trans-translation function of RpsA mediated through inhibition of tmRNA binding to RpsA.
  • Trans-translation is to rescue stalled ribosome facilitate the removal of deleterious partially translated proteins by adding a tmRNA encoded peptide tag recognized by proteases for degradation during stress conditions for cell survival. Trans-translation is dispensable during normal growth but becomes critical under stress conditions when bacteria stop growing, and POA inhibition of trans-translation is likely to affect the survival of non-growing persisters. Our study validates the trans-translation which is present in all bacteria but absent in eukaryotes as a critical target for developing persister drugs not only for TB but perhaps for other persistent bacterial pathogens.
Zhenkun Ma Video Presentation Discovery of a new generation pyrazinamide Zhenkun Ma

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  • Pyrazinamide plays an essential role in current and future TB treatment regimens for shortening therapy.
  • A new generation of pyrazinamide that is active against pyrazinamide resistance and has a wider therapeutic window without the need for weight banding is needed.
  • A variety of novel biological assays in both enzyme and whole-cell level are needed to ensure such compound can be identified.
Next Steps
Overview of Planned NIH Sponsored PZA Workshop scheduled for Spring 2012 Richard Hafner

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Background

Pyrazinamide (PZA) has potent sterilizing activity and is a highly important drug in current anti-tuberculosis (TB) combination therapy. Unfortunately, while PZA resistant TB has been increasing worldwide, rapid and reliable diagnostic tools for the detection of PZA-resistant TB are still unavailable. This presents a major barrier for treatment, especially for multi-drug resistant (MDR) and extensively drug resistant (XDR) disease. PZA is the least understood anti-TB drug due to its complex mechanisms of action and obstacles in establishing animal models for PZA testing.

Purpose

  1. To review current PZA use and historical data as well as the resistance patterns and clinical correlations in different regions; to address and identify the most promising research approaches for developing accurate and feasible PZA susceptibility testing. Mechanisms of action, how to determine optimal use in new combinations for drug-susceptible and -resistant TB, novel treatment strategies, and approaches to improve activity and overcome toxicity and resistance will also be discussed.
  2. To bring awareness to the scientific community regarding these research areas and to help identify the best approaches, strategies, and models for future advances in PZA research.
  3. To discuss future directions on PZA research and establish joint efforts and partnerships between government, industry, academia, and non-profit organizations.

Who should participate in the workshop

The majority of the invitees will be basic and clinical researchers from the US and the countries that have a high TB burden. Ideally, this meeting will also include scientists from related areas of research, e.g., geneticists, microbiologists, and immunologists who have not previously conducted PZA research or any TB research.

Core planning committee members

Under the auspice of the Federal TB Task Force, NIAID TB Clinical Research Team: Richard Hafner, Jing Bao; CDC: Michael Iademarco and Bonnie Plikaytis; and FDA: TBD; and from academia, JHU: TBD; Interested USG Parties: CDC, DTBE (Clinical Research Branch, Laboratory Branch); FDA (list)

Anticipated Outcome

  1. Networking and discussions from the meeting are expected to result in knowledge exchange and foster further collaborations and joint research projects to advance the field.
  2. A meeting report summarizing the state of the art in research on PZA and recommendations for future research will be composed. This report will be submitted for scientific publication to increase awareness in the scientific community and promote cutting-edge research.
  3. The research priorities identified from this meeting may serve as a source for the development of a a) Program Announcement or another type of NIH FOA to support needed research and b) refined CDC and FDA research agenda.

Number of Participants: 70 to 100 people will be invited to participate in this meeting.

Proposed date: FY 2012, Spring 2012

Download the Next Steps Plan [96kb]

Speakers & Attendees

Ammerman, Nicole

Center for Tuberculosis Research
Dept. of Medicine, Div. of Infectious Diseases
Johns Hopkins School of Medicine

Bao, Jing

Tuberculosis Clinical Research Team
Division of AIDS, NIAID, NIH, DHHS

Baughn, Anthony

Department of Microbiology
University of Minnesota

Bishai, William

Center for Tuberculosis Research
Director, KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH)
Dept. of Medicine, Div. of Infectious Diseases
Johns Hopkins School of Medicine

Blanchard, John

Albert Einstein College of Medicine

Boshoff, Helena

Tuberculosis Research Section, LCID, NIAID, NIH, DHHS

Deininger, David

Vertex Pharmaceuticals Incorporated

Farhat, Maha

Department of Pulmonary and Critical Care Medicine, Massachusetts General Hospital

Grosset, Jacques

Center for Tuberculosis Research
Dept. of Medicine, Div. of Infectious Diseases
Johns Hopkins School of Medicine

Hafner, Richard

Division of AIDS, NIAID, NIH, DHHS

Jacobs, William

Department of Microbiology & Immunology
Albert Einstein College of Medicine
Michael F. Price Center

Lacourciere, Karen

Respiratory Disease Branch, NIAID, NIH, DHHS

Laughon, Barbara

Division of Microbiology and Infectious Diseases, NIAID, NIH

Lenaerts, Anne

Department of Microbiology, Immunology and Pathology
Colorado State University

Locher, Christopher

Vertex Pharmaceuticals Incorporated

Ma, Zhenkun

TB Alliance

Makhene, Mamodikoe

Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS

Mendez, Susana

Baker Institute for Animal Health
Cornell University

Nuermberger, Eric

Dept. of Medicine, Div. of Infectious Diseases
Johns Hopkins School of Medicine

Russell, David

Microbiology and Immunology
College of Veterinary Medicine
Cornell University

Scott, Cherise

Working Group on New TB Drugs

Sizemore, Christine

Respiratory Disease Branch, NIAID, NIH, DHHS

Spigelman, Mel

TB Alliance

Vilcheze, Catherine

Department of Microbiology & Immunology
Albert Einstein College of Medicine

Welch, John

Chemistry
College of Arts and Sciences
University of Albany

Zhang, Ying

Molecular Microbiology and Immunology
Johns Hopkins Bloomberg School of Public Health

Zimhony, Oren

Division of Infectious Diseases
Kaplan Medical Center Rehovot
Hebrew University and Hadassah

Downloads

For more information regarding the Next Steps Plan, please contact:
Richard Hafner or Jing Bao